JPH0566113B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to L-glutamic acid, L-lysine, and L-glutamic acid, which are widely used in seasonings, feed additives, medicines, etc.
-Glutamine, L-arginine, L-phenylalanine, L-threonine, L-isoleucine, L
- Histidine, L-valine, L-serine, L-ornithine, L-citrulline, L-tyrosine, L-
The present invention relates to a method for producing L-amino acids such as tryptophan and L-leucine, and 5'-inosinic acid. (Prior art) L-glutamic acid, L-lysine, L-glutamine, L-arginine, L-phenylalanine, L-threonine, L-isoleucine, L-histidine, L-proline, L-valine, L-serine, L-ornithine, L-citrulline, L-tyrosine, L-tryptophan and L
- L-amino acids such as leucine and 5'-inosinic acid are industrially produced by fermentation methods. (Problems to be Solved by the Invention) The problems to be solved by the present invention are to develop a method for industrially producing L-amino acids or 5'-inosine at a lower cost by fermentation. (Means for Solving the Problems) As a result of extensive research into improving the yield of L-amino acid or 5'-inosinic acid fermentation as mentioned above, the present inventors found that N-methylglycine (hereinafter referred to as
), N,N-dimethylglycine (abbreviated as NG), N,N-dimethylglycine (hereinafter referred to as
(abbreviated as DMG), N,N,N-trimethylglycine (hereinafter abbreviated as TMG) and [2-hydroxyethyl]trimethylammonium (hereinafter abbreviated as TMG)
It has been found that the yield of the target L-amino acid or nucleic acid can be significantly improved by adding a certain amount of one or more substances selected from the group consisting of (abbreviated as HETMA) to a medium and fermenting the medium. The present invention was completed based on the above discovery in order to develop a method for industrially producing L-amino acids or 5'-inosinic acid that is cheaper than conventional methods. That is, the present invention
- Cultivate a microorganism capable of producing amino acids or 5'-inosinic acid in a medium containing one or more substances selected from the group consisting of NG, DMG, TMG, and HETMA, and The present invention relates to a method for producing L-amino acid or 5'-inosinic acid by a fermentation method, which is characterized by producing acid in a medium and collecting the acid. In L-amino acid fermentation or 5'-inosinic acid fermentation, these MG, DMG, TMG, and HETMA are added to a medium containing carbohydrates and other main carbon sources to perform a wide range of L-amino acid fermentation or 5'-inosinic acid fermentation. There is no knowledge that the yield has been improved. L with a small amount of Pseudomonas or Escherichia
- L-serine fermentation from betaine (Japanese Patent Application Laid-Open No. 1983-
134890) and L-amino acid fermentation using choline as the main carbon source using Pseudomonas sp.
154594) is known. However, in L-serine fermentation from betaine, betaine has been identified as a serine precursor, and from this fact it is not easy to infer a general increase in yield in L-amino acid fermentation.
Furthermore, even in L-amino acid fermentation using choline as the main carbon source, choline is the only main carbon source, and the yield of L-amino acids is small. On the other hand, L-
It is known that the fermentation yield of amino acids or 5'-inosinic acid can be significantly improved (Japanese Patent Application Laid-open No. 1986-1999).
216593), but there is no finding that the fermentation yield of L-amino acids or 5'-inosinic acid was significantly improved by further adding MG, DMG, TMG, or HETMA to a culture medium supplemented with stetsufene waste solution. The present invention is based on a novel discovery that makes it possible to greatly increase the yield of L-amino acid fermentation or 5'-inosinic acid using various main raw materials, and is extremely useful industrially. L-amino acids as used in the present invention include L-glutamic acid, L-lysine, L-glutamine, L-arginine, L-phenylalanine, L-threonine, and
-isoleucine, L-histidine, L-proline, L-valine, L-serine, L-ornithine,
L-amino acids such as L-citrulline, L-tyrosine, L-tryptophan, and L-leucine, and the method of the present invention can be used if L-amino acids other than those exemplified here are produced by fermentation. It is possible. The L-amino acid producing bacteria used in the present invention are the above-mentioned L-amino acid producing bacteria.
- Any microorganism may be used as long as it produces amino acids. in particular

[Table] Flavum

[Table] Glutamicum

[Table] Glycinophyllum

[Table] Glutamicum

[Table] etc. are used. The L-amino acid or 5'-inosinic acid production basic medium used in the present invention includes an L-amino acid production medium suitable for conventionally known L-amino acid producing bacteria and a production medium suitable for 5'-inosinic acid fermentation. Medium is available. In more detail, the main carbon sources include glucose, sucrose, fructose, maltose, sweet potato, molasses, sugar beet molasses, sugar beet molasses, raw sugar, starch saccharification liquid, cellulose saccharification liquid, pulp saccharification liquid, and lactose. Carbohydrates such as, fatty acids such as acetic acid, propenoic acid, benzoic acid, organic acids such as pyruvic acid, citric acid, succinic acid, fumaric acid, phosphoric acid, alcohols such as ethyl alcohol, butyl alcohol, etc. Or they can be used in combination. Furthermore, precursors of the biosynthetic pathway of the desired L-amino acid can also be used. Nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, urea, liquid ammonia,
Ammonia water, amino acid mixtures that are organic nitrogen compounds, corn steep liquor, casamino acids,
Yeast extract, soybean hydrolyzate, peptone, meat extract, etc. can be used. Inorganic salts include phosphates, magnesium salts, calcium salts, potassium salts, sodium salts, iron salts, manganese salts, zinc salts,
Use copper salts and other trace metals as necessary.
In addition, vitamins such as biotin and thiamine are used as necessary. The culture conditions in the present invention are the same as those in normal amino acid fermentation, and the culture temperature differs slightly depending on the target L-amino acid and the strain used.
A temperature of -40°C is preferred, and a temperature of 28 to 37°C is particularly suitable. PH
Better results can be obtained by controlling around neutrality during culture. The culture period for culturing under aerobic conditions such as aeration, stirring, and shaking culture is usually 1 to 7 days, but the period can be further extended by continuous culture or the like. Each amino acid is isolated from the fermentation broth by ion exchange resin treatment or other known methods. MG (CH ₃ NHCH ₂ COOH) used in the present invention,
DMG ((CH ₃ ) ₂ NCH ₂ COOH), TMG
((CH ₃ ) ₃ N ⁺ CH ₂ COO ^- or (OH) ^-
( _CH3 ) _3N ⁺ _CH2COOH ), HETMA((OH) ^-
(CH ₃ ) ₃ N ⁺ CH ₂ CH ₂ OH) and their salts are
It is available at low cost and in large quantities, not only as a reagent but also as an industrial product. MG is known as sarcosine, and is naturally present in flowers such as Hanago moss.It can be synthesized by a condensation reaction between methylamine and chloroacetic acid, or by Stretzker's amino acid synthesis method.
DMG is obtained by reacting chloroacetic acid with dimethylamine. TMG is known as glycine betaine, is abundant in sugar beets, cotton seeds, etc., and is synthesized by the reaction of chloroacetic acid and trimethylamine. HETMA is known as choline and is abundant in radish leaves, etc., and is synthesized by reacting trimethylamine with a concentrated aqueous solution of ethylene oxide. In addition, these various salts are commercialized; for example, sodium salt for MG, hydrochloride for DMG and TMG,
Regarding HETMA, chloride salt, hydrogen tartrate, dihydrogen citrate, gluconate, etc. are industrially produced and easily available. MG, DMG, TMG or
HETMA may be their salts or may be replaced with natural products containing them. The method of using MG, DMG, TMG, and HETMA in the present invention is to include them alone or in combination of two or more in a fermentation medium at a concentration of 0.01 to 5%, preferably 0.05 to 2.0%. That's fine. These may be added to the fermentation medium in advance to start the culture, or may be added once, several times, or continuously during the culture. Alternatively, it may be added to the seed culture and brought into the main fermentation. Example 1 Glucose 3g/dlKH ₂ PO ₄ 0.1g/dl
MgSO ₄・7H ₂ O0.05g/dlFeSO ₄・7H ₂ O1mg/dl
MnSO ₄・4H ₂ O1mg/dl urea 0.5g/dl bition
A seed medium containing 30 μg/100 μg of thiamine hydrochloride/50 mg/dl of soybean protein hydrolyzate as total urea was adjusted to pH 7.5, and 50 ml of the mixture was placed in a 500 ml shoulder flask and sterilized by heating. Brevibacterium
Lactofermentum ATCC 13869 inoculation 31.5
The cells were cultured with shaking for 16 hours while being maintained at ℃. On the other hand, as a glutamate production medium, cane molasses (as sugar) 8.0g/dl, KH ₂ PO ₄ 0.2g/dl,
MgSO ₄・7H ₂ O0.1g/dl, FeSO ₄・4H ₂ O2.0mg/
dl, thiamine hydrochloride 200μg/soybean protein hydrolyzate (as total nitrogen) 30mg/dl, Stetsufuen concentrated waste liquid (total nitrogen concentration 2.0g/dl) total nitrogen concentration 200mg/dl, and TMG 0.01 to 5g/dl After adjusting the pH to 7.8, 285 ml of each medium was dispensed into small 1.0 volume fermenters, and the medium was heated at 115°C for 10 minutes to sterilize it. Seed culture solution 15 obtained by culturing in each medium in advance
ml each was inoculated and culture with aeration and stirring was started at 31.5°C. During the culture period, the pH of the culture solution was maintained at PH7.8 using ammonia gas, and pre-sterilized cane molasses was added to adjust the sugar content in the culture solution to 2 to 4 g/dl in terms of sucrose. In addition, polyoxyethylene sorbitan monopalmitate was added at an appropriate time during the culture, and the sugar supply was stopped when the sugar consumption rate had substantially decreased.
Culture was terminated after 28 to 40 hours. The amount of L-glutamic acid accumulated in each culture solution was measured using a conventional method, and the yield relative to sugar was determined. The results are shown in Table 1.

[Table] Example 2 Glucose 5g/dl, (NH ₄ ) ₂ SO ₄ 0.2g/dl,
Urea 0.2g/dl, KH ₂ PO ₄ 0.15g/dl, MgSO ₄・
A seed medium containing 0.04 g/dl of 7H ₂ O, 1 mg/dl of FeSO ₄ 7H ₂ O, 100 μg/thiamine HCl, 3 μg/dl of biotin, and a total nitrogen content of 30 mg/dl of soybean protein acid decomposition solution was adjusted to pH 7.0. 50 ml of the mixture was placed in a 500 ml shoulder flask and sterilized by heating. This includes the glutamine-fermenting bacterium Brevibacterium flavum FERM BP.
-662 was inoculated and shaken for 12 hours while keeping at 31.5°C. On the other hand, glucose 13g/dl, KH ₂ PO ₄ 0.25g/
dl, MgSO ₄・7H ₂ O40mg/dl, (NH ₄ ) ₂ SO ₄ 1.5
g/dl, Na ₂ SO ₄ 1.5g/dl, FeSO ₄・7H ₂ O0.1
mg/dl, biotin 3.5 μg/, thiamine/hydrochloride 350 μg/, HETMA 0.01 to 5 to make the total nitrogen concentration of the concentrated stetsufen waste liquid by-produced in the stetsufen process of the beet sugar factory to 500 mg/dl.
1 g/dl added and 285 ml of medium adjusted to pH 6.5.
It was sterilized by placing it in a container. Each of these was inoculated with 15 ml of the above seed mother culture solution. Cultivation was carried out at 31.5°C while maintaining the pH at 6.5 to 6.0 with NH ₃ gas and stirring until the residual glucose was 0.5 g/dl.
I went until I became The yield of L-glutamine based on sugar weight is shown in Table 2. Table 2 HETMA addition amount L-glutamine yield 0g/dl 41.0% 0.01 41.5 0.05 42.0 0.25 42.5 0.5 43.0 1.0 44.0 2.0 43.0 3.0 41.5 5.0 40.0 The yield was higher than the control in the plots with HETMA0.01~3g/dl added. improved was. Culture solution 1 was obtained from the group containing 1.0 g/dl of HETMA, and the bacterial cells were removed by centrifugation, and the supernatant was passed through a strongly acidic ion exchange resin "Diaion" SK-1B (NH ₄ ⁺ type). Ta. After washing the resin with water, it was eluted with 2N-NH ₄ OH, and the eluate was then concentrated to obtain crude crystals of L-glutamine.
35.9g was obtained. Example 3 Glucose 13g/dl, (NH ₄ ) ₂ SO ₄ 6g/dl,
KH ₂ PO ₄ 0.1g/dl, MgSO ₄・7H ₂ O0.04g/dl,
FeSO ₄・7H ₂ O1mg/dl, MnSO ₄・4H ₂ O1mg/dl,
Thiamine/hydrochloride 100μg/, biotin 50μ
HETMA0.01-3.0g/dl was added to the concentrated stetsufen waste liquid produced as a by-product in the stetsufen process at a beet sugar factory with a total nitrogen concentration of 500mg/dl, and calcium carbonate 5g/dl (separately sterilized) was added. The containing medium was adjusted to pH 7.0, and 20 ml of it was placed in a 500 ml shoulder flask and sterilized by heating. This includes Brevibacterium flavum NRRLB.
-12235 was inoculated with one platinum loop, and the temperature was kept at 31.5℃.
Shake for days. The yield of L-arginine based on sugar weight is shown in Table 3. Table 3 HETMA addition amount L-arginine yield 0 g/dl 30.5% 0.01 31.0 0.05 31.3 0.25 32.0 0.5 33.0 1.0 32.0 2.0 31.0 3.0 29.5 Culture solution 1 of HETMA 0.5 g/dl addition group was obtained.
From this, bacterial cells and other substances are removed by centrifugation, and the supernatant is collected using a weakly acidic ion exchange resin "Amberlite" C-50.
(NH ₄ ⁺ form). 2N− after washing the resin with water
L-arginine was eluted with NH ₄ OH, the eluate was concentrated, and crude crystals of L-arginine were obtained.
30.5g was obtained. Example 4 0.01 to 3 g/dl of DMG was added to a medium containing glucose as a carbon source and having the composition shown in Table 4.
- After adjusting the isoleucine production medium and adjusting the pH to 7.2, 20 ml each was dispensed into 500 ml shaking flasks and heated at 115° C. for 10 minutes to sterilize.

[Table] Each medium was inoculated with Brevibacterium flavum FERM BP-759 obtained by culturing in advance, and cultured with shaking at 31.5°C until glucose disappeared. Table 5 shows the yield of L-isoleucine based on sugar weight. Table 5 HDMG addition amount L-isoleucine yield 0 g/dl 12.0% 0.01 12.5 0.05 12.7 0.25 13.0 0.5 13.3 1.0 13.5 2.0 13.0 3.0 11.5 Example 5 0.01 TMG was added to the medium shown in Table 6 containing glucose as a carbon source. A medium for L-valine production was prepared by adding ~3 g/dl, and after adjusting the pH to 7.0,
The mixture was dispensed into 20 ml shake flasks and sterilized by heating at 115°C for 10 minutes.

[Table] Brevibacterium lactofermentum FERM BP obtained by culturing in each medium in advance
-1896 was inoculated and cultured with shaking at 31.5°C until glucose was consumed. Table 7 shows the yield of L-valine based on sugar. Table 7 TMG addition amount L-valine yield 0 g/dl 12.0% 0.01 12.5 0.05 13.0 0.25 13.5 0.5 14.0 1.0 13.5 2.0 12.5 3.0 11.5 Example 6 50 ml of the seed medium having the composition shown in Table 8 was placed in a 500 ml shaking flask. After dispensing and heat sterilizing at 110℃ for 5 minutes, remove Brevibacterium flavum.
ATCC21475 was inoculated and cultured with shaking at 31.5°C for 20 hours to obtain a seed culture. On the other hand, L- shown in Table 4
20 ml of the medium for lysine production was dispensed into Sakaguchi flasks and sterilized. 1.5 g of calcium carbonate, which had been dry-heat sterilized in advance, was added to each of these, and then 1 ml of the above seed culture solution was inoculated to each, and cultured at 31.5° C. with reciprocal shaking. 0, 0.01, 0.05, 0.25, 1.0, 2.0, 5.0 g/dl of TMG per medium at 24 hours of culture
The culture was continued. Generated L
- The amount of lysine accumulated and the yield are shown in Table 9.

【table】

[Table] Collect the cultured solution with TMG addition amount of 0.25 g/dl, remove the bacterial cells and calcium salts by centrifugation, and transfer the supernatant solution 1 to a strongly acidic ion exchange resin ("Amberlite" IR-120CH type). ) to adsorb L-lysine. Next, the adsorbed L-lysine was eluted with 3% aqueous ammonia, and the eluate was concentrated under reduced pressure. After adding hydrochloric acid to the concentrated solution, it was cooled, and L-lysine was precipitated as L-lysine hydrochloride second hydrate to obtain 36.5 g of crystals. Example 7 Glucose 3g/dl, (NH ₄ ) ₂ SO ₄ 0.2g/dl,
Urea 0.2g/dl, KH ₂ PO ₄ 0.15g/dl, MgSO ₄・
_7H2O0.04g /dl, _FeSO4・_7H2O1mg /dl, Thiamine・HCl100μg/, Biotin 300μg/,
140mg/dl of soybean protein acid hydrolyzate as total nitrogen
Adjust the seed culture medium containing PH7.0 to 500ml
The mixture was placed in a ml shoulder flask and sterilized by heating. Brevibacterium flavum ATCC21269 and Estherichia coli FERMBP-1483 (AJ 11334) were each inoculated, and cultured with shaking while maintained at 31.5°C for 12 hours to obtain a seed mother culture. On the other hand, glucose 13g/dl, KH ₂ PO ₄ 0.25g/
dl, MgSO ₄・7H ₂ O40mg/dl, (NH ₄ ) ₂ SO ₄ 2.0
g/dl, MnSO ₄・4H ₂ O1mg/dl, FeSO ₄・
7H ₂ O 1mg/dl, L-isoleucine 40mg/dl, biotin 50μg/dl, thiamine/HCl 5μg/dl,
32mg/dl of soybean protein acid hydrolyzate as total nitrogen,
A medium adjusted to pH 6.5 containing 200 mg/dl of Stetsufuen waste solution (as total nitrogen) and TMG 0.01 to 5
20 ml of the medium to which g/dl had been added was dispensed into a 500 ml flask with a shoulder and steam sterilized at 110°C for 10 minutes. Add to this 2 g of calcium carbonate, which has been dry heat sterilized in advance.
to each, and then add 1 ml of the above seed culture solution.
and cultured at 31.5°C. Table 10 shows the sugar yield of L-threonine obtained in the culture solution.

[Table] TMG addition to Brevibacterium flavum
Culture solution 1 of 0.25 g/dl was obtained, bacterial cells and calcium carbonate were removed from the culture solution by centrifugation, and 500 ml of the solution was treated with strongly acidic cation exchange resin ("Amberlight" IR-120 (H ⁺ type)). ) column. Elution was performed with 3% aqueous ammonia, and the amino acid fractions were collected, desalted and decolorized, and concentrated under reduced pressure. After adding alcohol and storing under cooling, the resulting crystals were collected and dried, resulting in 7.6 g of threonine crystals with a purity of 98% or higher. Example 8 In the L-histidine production medium shown in Table 11
500ml after adding MG0.01~3g/dl and adjusting the pH to 7.2
20 ml of each was dispensed into shaking flasks and sterilized by heating at 115°C for 10 minutes.

[Table] Brevibacterium flavum obtained by culturing on bouillon slant in advance on each medium.
ATCC 21406 was inoculated and shaken at 31.5°C until glucose disappeared. Table 12 shows the yield of L-histidine based on sugar. Table 12 MG addition amount L-histidine yield 0 10.0 0.01 10.5 0.025 11.0 0.5 11.5 1.0 12.0 2.0 11.0 3.0 9.5 Remove the bacterial cells and calcium carbonate from the culture solution 1 of the MG0.5g/dl addition group and remove the supernatant with strongly acidic ions. It was passed through an exchange resin ("Amberlite" IR-120 (H ⁺ type)) to adsorb L-histidine. then 3
% aqueous ammonia was used to elute the adsorbed L-histidine, and the eluate was concentrated under reduced pressure. When the concentrated liquid was cooled and left to stand, L-histidine crystals were precipitated. The crystals were dried to obtain 7.1 g. Example 9 A medium for L-phenylalanine production was prepared by adding DMG 0.01 to 3.0 g/dl to a medium containing seuculose as a carbon source and having the composition shown in Table 13,
After adjusting the pH to 7.0, add each to a 500ml shake flask.
It was dispensed into 20 ml volumes and heated at 115°C for 10 minutes to sterilize it.

[Table] Each medium was inoculated with Brevibacterium lactofermentum ATCC 21420, which had been previously cultured on a bouillon slant, and cultured with shaking at 31.5°C until glucose disappeared. The yield of phenylalanine based on sugar was as shown in Table 14. Table 14 DMG addition amount L-phenylalanine yield 0 g/dl 9.0% 0.01 9.5 0.02 10.5 0.5 11.0 1.0 11.5 2.0 11.0 3.0 10.5 Example 10 Add TMG to a medium containing glucose as a carbon source and having the composition shown in Table 15. .01~5g/dl added L
- A tryptophan production medium was prepared, and after adjusting the pH to 7.0, 20 ml of each was dispensed into 500 ml shaking flasks and sterilized by heating at 115° C. for 10 minutes.

[Table] Bacillus subtilis FERM BP-208, which had been cultured in advance, was inoculated into each medium and incubated at 30°C.
The cells were cultured with shaking until glucose disappeared. L
-The yield of tryptophan based on sugar is shown in Table 16. Table 16 TMG addition amount L-tryptophan yield 0 g/dl 5.0% 0.01 6.0 0.02 6.5 0.5 7.0 1.0 7.5 2.0 6.5 3.0 6.0 5.0 5.5 Example 11 Addition of DMG to a medium containing glucose as a carbon source and having the composition shown in Table 17 Add .01~3g/dl L
-Prepare a medium for serine production and adjust the pH to 500 ml.
Dispense 20 ml each into ml shake flasks,

【table】

[Table] Corynebacterium glycinophyllum FERM-P obtained by culturing in each medium in advance
1687 was inoculated and cultured with shaking at 34°C. Table 18 shows the molar yield of consumed glycine for L-serine. Table 18 DMG addition amount L-serine yield 0 g/dl 36.0% 0.01 37.0 0.05 38.0 1.0 38.5 2.0 37.5 3.0 35.0 Example 12 Add TMG 0.1 to 5 g/dl to the medium with the composition shown in Table 19
Add and dispense 20 ml each into a 500 ml shoulder flask.
Heat sterilized at ℃ for 10 minutes. Corynebacterium equii AJ 11749 (FERM-P6261) was added to this medium at 20g/glucose/10g/yeast extract/10g/peptone/5g/salt/20g/agar/(PH
The cells obtained by culturing at 31.5℃ for 18 hours on a slant medium with the composition of 7.2) were inoculated with 3 platinum loops, and after culturing at 34℃ for 2 days, glucose 50g/, inosine 10
g/ was added and cultured with shaking for an additional 3 days. The amount of 5′-inosinic acid accumulated in the culture solution was
Table 20 shows this.

【table】

[Table] Table 20 5'-inosinic acid accumulation TMG addition amount (5'-IMP・2Na・7.5H ₂ O) 0g/dl 28 0.1 28.5 0.5 29 1.0 30 2.0 29.5 3.0 29 5.0 27 Example 13 Glucose 3g /dl, KH ₂ PO ₄ 0.1g/dl,
MgSO ₄・7H ₂ O0.04g/dl, FeSO ₄・7H ₂ O0.001
g/dl, MnSO ₄・4H ₂ O0.001g/dl, urea 0.3
g/dl, 60 as total nitrogen for soybean protein acid hydrolyzate
mg/dl, thiamine/HCl30μg/dl, biotin
The seed medium containing 20 μg/dl was adjusted to pH 6.0, and the
50ml was placed in a 500ml shoulder flask and sterilized by heating. This was inoculated with the strain Brevibacterium lactofermentum FERM-P 5936 (AJ 11678), and cultured with shaking while maintaining the temperature at 31.5°C for 13 hours. On the other hand, glucose 13g/dl, KH ₂ PO ₄ 0.1g/
dl, MgSO ₄・7H ₂ O0.04g/dl, FeSO ₄・
_7H2O0.001g /dl, _MnSO4・_4H2O0.001g /dl,
Ammonium sulfate 4.0g/dl, soybean protein acid hydrolyzate as total nitrogen 65mg/dl, thiamine/HCl 20μg/dl,
A medium adjusted to pH 7.0 containing 10 μg/dl of biotin and 70 mg/dl of L-arginine and 0.01 to 3 DMG
20 ml of each medium supplemented with g/dl was dispensed into 500 ml shouldered flasks and sterilized by heating at 115° C. for 10 minutes. 2 g of calcium carbonate, which had been dry heat sterilized in advance, was added to each of these, and then 1 g of the above seed culture solution was added.
ml each was added and cultured at 31.5°C. The yield of L-ornithine relative to sugar at the end of the culture is shown in Table 21. Table 21 DMG addition amount L-ornithine yield 0g/dl 24.5% 0.01 26.0 0.05 27.5 1.0 29.0 2.0 27.0 3.0 23.0 Example 14 Glucose 3g/dl, KH ₂ PO ₄ 0.1g/dl,
MgSO ₄・7H ₂ O0.04g/dl, FeSO ₄・7H ₂ O0.001
g/dl, MnSO ₄・4H ₂ O0.001g/dl, urea 0.3
g/dl, 60 as total nitrogen for soybean protein acid hydrolyzate
mg/dl, thiamine/HCl30μg/dl, biotin
The seed medium containing 20 μg/dl was adjusted to pH 6.0, and the
50ml was placed in a 500ml shoulder flask and sterilized by heating. In this, the strain Corynebacterium glutamicum
ATCC 13232 was inoculated and cultured with shaking for 13 hours while maintaining the temperature at 31.5°C. On the other hand, glucose 13g/dl, KH ₂ PO ₄ 0.1g/
dl, MgSO ₄・7H ₂ O0.04g/dl, FeSO ₄・
_7H2O0.001g /dl, _MnSO4・_4H2O0.001g /dl,
Ammonium sulfate 4.0g/dl, soybean protein acid hydrolyzate as total nitrogen 65mg/dl, thiamine/HCl 20μg/dl,
A medium adjusted to pH 7.0 containing biotin 10 μg/dl and L-homoserine 70 mg/dl and DMG 0.01-3
20 ml of each medium supplemented with g/dl was dispensed into 500 ml shouldered flasks and sterilized by heating at 115° C. for 10 minutes. 2 g of calcium carbonate, which had been dry heat sterilized in advance, was added to each of these, and then 1 g of the above seed culture solution was added.
ml each was added and cultured at 31.5°C. The yield of L-ornithine relative to sugar at the end of the culture is shown in Table 22. Table 22 DMG addition amount L-ornithine yield 0 g/dl 24.5% 0.01 26.0 0.05 27.5 1.0 29.0 2.0 27.0 3.0 23.0 Example 15 Glucose 3 g/dl, KH ₂ PO ₄ 0.1 g/dl,
MgSO ₄・7H ₂ O0.04g/dl, FeSO ₄・7H ₂ O1.0
mg/dl, MnSO ₄・4H ₂ O1.0mg/dl, urea 0.3g/
dl, 60mg/total nitrogen of soybean protein acid hydrolyzate
dl, thiamine/HCl30γ/dl, biotin 20γ/dl
Adjust the seed culture medium containing PH6.0 to 500ml
The mixture was placed in a ml shoulder flask and sterilized by heating. Corynebacterium glutamicum FERM-P 5643
(AJ 11588) and cultured with shaking for 13 hours while maintaining at 31.5°C. On the other hand, glucose 13g/dl, KH ₂ PO ₄ 0.1g/
dl, MgSO ₄・7H ₂ O0.04g/dl, FeSO ₄・
7H ₂ O 1.0 mg/dl, MnSO ₄・4H ₂ O 1.0 mg/dl, ammonium chloride
3.0g/dl, 65mg/dl based on total nitrogen of soybean protein acid hydrolyzate, PH7.0 including thiamine/HCl20γ/dl, biotin 10γ/dl, L-arginine 70mg/dl
Cultures were carried out in the same manner as in Example 14 using a medium adjusted to 0.2 g/dl and a medium to which 0.01 to 3 g/dl of MG was added. The yield of L-citrulline relative to sugar at the end of the culture was as shown in Table 23. Table 23 MG addition amount L-citrulline yield 0 g/dl 13.5% 0.01 14.0 0.05 14.5 1.0 15.5 2.0 14.0 3.0 13.0 Example 16 Dispense 50 ml of the vertical mother medium having the composition shown in Table 24 into a 500 ml shaking flask. After heating and sterilizing at 110°C for 5 minutes, the strain Corynebacterium glutamicum FERM-P 5836 (AJ 11655) was inoculated at 31.5°C.
A seed culture solution was obtained by performing shaking culture for 12 hours. On the other hand, the L-tyrosine main fermentation medium in Table 25 and this
Each medium containing 0.01 to 3 g/dl of HETMA was cultured in the same manner as in Example 14. The amount of L-tyrosine at the end of the culture was as shown in Table 25.

[Table] Table 25 HETMA addition amount L-tyrosine yield 0g/dl 6.0% 0.01 6.5 0.03 8.0 0.5 9.0 1.0 8.2 2.0 7.5 3.0 6.0 Example 17 Glucose 3g/dl, KH ₂ PO ₄ 0.1g/dl, Urea 0.3 g/dl, MgSO ₄・7H ₂ O0.04g/dl,
FeSO ₄・7H ₂ O1mg/dl, MnSO ₄・4H ₂ O1mg/dl,
DL-methionine 40mg/dl, biotin 10μg/,
A seed medium containing 200 μg/dl of thiamine hydrochloride and 50 mg/dl of soybean protein acid hydrolyzate as total nitrogen was adjusted to pH 6, and 50 ml of it was placed in a 500 ml flask with a shoulder and heat sterilized at 120°C for 10 minutes. . This includes Brevibactenium flavum FERM BP−755
was inoculated and cultured with shaking at 31.5°C for 20 hours. On the other hand, sugar beet molasses (converted to glucose) 13g/
dl, KH ₂ PO ₄ 0.1g/dl, (NH ₄ ) ₂ SO ₄ 2.0g/dl,
MgSO ₄・7H ₂ O0.04g/dl, FeSO ₄・7H ₂ O1mg/
dl, MnSO ₄ 4H ₂ O1mg/dl, DL-methionine
A medium adjusted to PH7.0 containing 100mg/dl, biotin 50μg/, thiamine hydrochloride 300μg/, defatted soybean acid decomposition solution 50mg/dl, and HETMA0.01
Each medium containing ~3 g/dl was cultured in the same manner as in Example 14. The amount of L-leucine produced at the end of the culture was as shown in Table 26. Table 26 HETMA addition amount L-leucine yield 0g/dl 12.0% 0.01 13.2 0.5 14.2 1.0 15.0 2.0 14.0 3.0 12.5

Claims (1)

[Claims] 1 L belonging to the genus Brevibacterium, Corynebacterium, Eserichia or Bacillus
- Amino acid-producing bacteria or 5'-inosinic acid-producing bacteria belonging to the genus Corynebacterium are used as main carbon sources, such as cane molasses, sucrose, or glucose, and N
-Methylglycine, N,N-dimethylglycine,
Cultured in a medium containing one or more substances selected from the group consisting of N,N,N-trimethylglycine and [2-hydroxyethyl]trimethylammonium to produce L-amino acids or 5'-inosinic acid, 1. A method for producing L-amino acid or 5'-inosinic acid by a fermentation method, which comprises collecting .